Differential dynamic properties of scleroderma fibroblasts in response to perturbation of environmental stimuli.

Diseases are believed to arise from dysregulation of biological systems (pathways) perturbed by environmental triggers. Biological systems as a whole are not just the sum of their components, rather ever-changing, complex and dynamic systems over time in response to internal and external perturbation. In the past, biologists have mainly focused on studying either functions of isolated genes or steady-states of small biological pathways. However, it is systems dynamics that play an essential role in giving rise to cellular function/dysfunction which cause diseases, such as growth, differentiation, division and apoptosis. Biological phenomena of the entire organism are not only determined by steady-state characteristics of the biological systems, but also by intrinsic dynamic properties of biological systems, including stability, transient-response, and controllability, which determine how the systems maintain their functions and performance under a broad range of random internal and external perturbations. As a proof of principle, we examine signal transduction pathways and genetic regulatory pathways as biological systems. We employ widely used state-space equations in systems science to model biological systems, and use expectation-maximization (EM) algorithms and Kalman filter to estimate the parameters in the models. We apply the developed state-space models to human fibroblasts obtained from the autoimmune fibrosing disease, scleroderma, and then perform dynamic analysis of partial TGF-beta pathway in both normal and scleroderma fibroblasts stimulated by silica. We find that TGF-beta pathway under perturbation of silica shows significant differences in dynamic properties between normal and scleroderma fibroblasts. Our findings may open a new avenue in exploring the functions of cells and mechanism operative in disease development.

An mRNA-derived ncRNA targets and regulates the ribosome

Small non-protein-coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily, not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). All of the recently discovered regulatory ncRNAs that act on translation (e.g. microRNAs, siRNAs or antisense RNAs) target the mRNA rather than the ribosome. This is unexpected, given the central position the ribosome plays during gene expression. To investigate whether such a class of regulatory ncRNAs does exist we performed genomic screens for small ribosome-associated RNAs in various model organisms of all three domains [1,2]. Here we focus on the functional characterisation of an 18 nucleotide long ncRNA candidate derived from an open reading frame (ORF) of an annotated S. cerevisiae gene, which encodes a tRNA methyltransferase. Yeast cells lacking this tRNA methyltransferase showed clear growth defects in high salt containing media. Genetic analysis showed that the absence of the mRNA-derived ncRNA rather than the absence of the tRNA methyltransferase activity is responsible for the observed phenotype. Since we performed a screen for small ribosome-associated RNAs we examined the regulatory potential of the synthetic 18mer during translation in vitro and in vivo. Metabolic labeling experiments in the presence of the synthetic 18mer RNA revealed an inhibitory potential on the global protein biosynthesis rate. In vitro translation and northern blot analysis further strengthen the hypothesis, that this RNA is a ribosome-associated regulatory ncRNA. Our studies in pro- and eukaryotic model organisms reveal the ribosome as a novel target for small regulatory ncRNAs in all domains of life. Ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory ncRNAs.

When small non-coding RNAs meet the ribosome: Tuning the translational machinery

The functions of ribosomes in translation are complex and involve different types of activities critical for decoding the genetic code, linkage of amino acids via amide bonds to form polypeptide chains, as well as the release and proper targeting of the synthesized protein. Non-protein-coding RNAs (ncRNAs) have been recognized to be crucial in establishing regulatory networks.1 However all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. The main goal of this project is to identify potential novel ncRNAs that directly bind and possibly regulate the ribosome during protein biosynthesis. To address this question we applied various stress conditions to the archaeal model organism Haloferax volcanii and deep-sequenced the ribosome-associated small ncRNA interactome. In total we identified 6.250 ncRNA candidates. Significantly, we observed the emersed presence of tRNA-derived fragments (tRFs). These tRFs have been identified in all domains of life and represent a growing, yet functionally poorly understood, class of ncRNAs. Here we present evidence that tRFs from H. volcanii directly bind to ribosomes. In the presented genomic screen of the ribosome-associated RNome a 26 residue long fragment originating from the 5’ part of valine tRNA was by far the most abundant tRF. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. As a consequence of ribosome binding, Val-tRF reduces protein synthesis by interfering with peptidyl transferase activity. Therefore this tRF functions as ribosome-bound small ncRNA capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production.2 Currently we are investigating the binding site of this tRF on the 30S subunit in more detail.

Role of MicroRNA Modulation in the Interferon-α/Ribavirin Suppression of HIV-1 In Vivo.

BACKGROUND Interferon-α (IFN-α) treatment suppresses HIV-1 viremia and reduces the size of the HIV-1 latent reservoir. Therefore, investigation of the molecular and immunologic effects of IFN-α may provide insights that contribute to the development of novel prophylactic, therapeutic and curative strategies for HIV-1 infection. In this study, we hypothesized that microRNAs (miRNAs) contribute to the IFN-α-mediated suppression of HIV-1. To inform the development of novel miRNA-based antiretroviral strategies, we investigated the effects of exogenous IFN-α treatment on global miRNA expression profile, HIV-1 viremia, and potential regulatory networks between miRNAs and cell-intrinsic anti-HIV-1 host factors in vivo. METHODS Global miRNA expression was examined in longitudinal PBMC samples obtained from seven HIV/HCV-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated interferon-α/ribavirin therapy (IFN-α/RBV). We implemented novel hybrid computational-empirical approaches to characterize regulatory networks between miRNAs and anti-HIV-1 host restriction factors. RESULTS miR-422a was the only miRNA significantly modulated by IFN-α/RBV in vivo (p<0.0001, paired t test; FDR<0.037). Our interactome mapping revealed extensive regulatory involvement of miR-422a in p53-dependent apoptotic and pyroptotic pathways. Based on sequence homology and inverse expression relationships, 29 unique miRNAs may regulate anti-HIV-1 restriction factor expression in vivo. CONCLUSIONS The specific reduction of miR-422a is associated with exogenous IFN-α treatment, and likely contributes to the IFN-α suppression of HIV-1 through the enhancement of anti-HIV-1 restriction factor expression and regulation of genes involved in programmed cell death. Moreover, our regulatory network analysis presents additional candidate miRNAs that may be targeted to enhance anti-HIV-1 restriction factor expression in vivo.

Ribosome-associated ncRNAs (rancRNAs) An emerging class of translation regulators

Small non-protein-coding RNA (ncRNA) molecules represent major contributors to regulatory networks in controlling gene expression in a highly efficient manner. Most of the recently discovered regulatory ncRNAs acting on translation target the mRNA rather than the ribosome (e.g.: miRNAs, siRNAs, antisense RNAs). To address the question, whether ncRNA regulators exist that are capable of modulating the rate of protein production by directly interacting with the ribosome, we have analyzed the small ncRNA interactomes of ribosomes. Deep-sequencing analyses revealed thousands of putative rancRNAs in various model organisms (1,2). For a subset of these ncRNA candidates we have gathered experimental evidence that they associate with ribosomes in a stress-dependent manner and fine-tune the rate of protein biosynthesis (3,4). Many of the investigated rancRNAs appear to be processing products of larger functional RNAs, such as tRNAs (2,3), mRNAs (3), or snoRNAs (2). Post-transcriptional cleavage of RNA to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. Our data disclose the ribosome as target for small regulatory RNAs. rancRNAs are found in all domains of life and represent a prevalent but so far largely unexplored class of regulatory molecules (5). Ongoing work in our lab revealed first insight into rancRNA processing and mechanism of this emerging class of translation regulators.

Biology of tRNA halves in Trypanosoma brucei

Although T. brucei has to challenge tremendous environment changes, e.g. switch from the bloodstream form in mammalian hosts to the mid gut form present in tsetse flies, there is no evidence for differential regulation of RNA Pol II transcription. Instead, constitutive transcription appears to occur. This observation indicates that protein levels have to be regulated by post-transcriptional mechanisms. It has been shown that non-protein coding RNAs (ncRNAs) are crucial in regulatory networks (e.g. chromosome remodelling; RNA polymerase activity; mRNA turnover; etc.), but all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. This is unexpected, since the ribosome has a central role during gene expression and due to the assumption that the primordial translation system most likely received direct regulatory input from small molecules including ncRNA cofactors. In our lab, it has been discovered that ncRNAs are able to directly bind to the ribosome, therefore influencing the translation rate in Haloferax volcanii and Saccharomyces cerevisiae. In order to extend this idea of ribosome-binding ncRNAs in mammalian parasites, we want to investigate this mechanism in T. brucei. Accordingly, we performed a genomic screen for small ribosome-associated RNAs followed by functional analyses of possible candidates. With the help of this genomic screen, we found tRNAs that are alternated and tRNA halves that are differentially expressed upon nutritional stress.

Biology of tRNA halves in Trypanosoma brucei

Although T. brucei has to challenge tremendous environment changes, e.g. switch from the bloodstream form in mammalian hosts to the mid gut form present in tsetse flies, there is no evidence for differential regulation of RNA Pol II transcription. Instead, constitutive transcription appears to occur. This observation indicates that protein levels have to be regulated by post-transcriptional mechanisms. It has been shown that non-protein coding RNAs (ncRNAs) are crucial in regulatory networks (e.g. chromosome remodelling; RNA polymerase activity; mRNA turnover; etc.), but all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. This is unexpected, since the ribosome has a central role during gene expression and due to the assumption that the primordial translation system most likely received direct regulatory input from small molecules including ncRNA cofactors. In our lab, it has been discovered that ncRNAs are able to directly bind to the ribosome, therefore influencing the translation rate in Haloferax volcanii and Saccharomyces cerevisiae. In order to extend this idea of ribosome-binding ncRNAs in mammalian parasites, we want to investigate this mechanism in T. brucei. Accordingly, we performed a genomic screen for small ribosome-associated RNAs followed by functional analyses of possible candidates. With the help of this genomic screen, we found tRNAs that are alternated and tRNA halves that are differentially expressed upon nutritional stress.

Systems Biology Approaches to Probe Gene Regulation in Bacteria

Mechanisms that allow pathogens to colonize the host are not the product of isolated genes, but instead emerge from the concerted operation of regulatory networks. Therefore, identifying components and the systemic behavior of networks is necessary to a better understanding of gene regulation and pathogenesis. To this end, I have developed systems biology approaches to study transcriptional and post-transcriptional gene regulation in bacteria, with an emphasis in the human pathogen Mycobacterium tuberculosis (Mtb). First, I developed a network response method to identify parts of the Mtb global transcriptional regulatory network utilized by the pathogen to counteract phagosomal stresses and survive within resting macrophages. As a result, the method unveiled transcriptional regulators and associated regulons utilized by Mtb to establish a successful infection of macrophages throughout the first 14 days of infection. Additionally, this network-based analysis identified the production of Fe-S proteins coupled to lipid metabolism through the alkane hydroxylase complex as a possible strategy employed by Mtb to survive in the host. Second, I developed a network inference method to infer the small non-coding RNA (sRNA) regulatory network in Mtb. The method identifies sRNA-mRNA interactions by integrating a priori knowledge of possible binding sites with structure-driven identification of binding sites. The reconstructed network was useful to predict functional roles for the multitude of sRNAs recently discovered in the pathogen, being that several sRNAs were postulated to be involved in virulence-related processes. Finally, I applied a combined experimental and computational approach to study post-transcriptional repression mediated by small non-coding RNAs in bacteria. Specifically, a probabilistic ranking methodology termed rank-conciliation was developed to infer sRNA-mRNA interactions based on multiple types of data. The method was shown to improve target prediction in Escherichia coli, and therefore is useful to prioritize candidate targets for experimental validation.

REGULATION OF ALTERNATIVE CARBON METABOLISM IN CANDIDA ALBICANS

Candida albicans is the most important fungal pathogen of humans. Transcript profiling studies show that upon phagocytosis by macrophages, C. albicans undergoes a massive metabolic reorganization activating genes involved in alternative carbon metabolism, including the glyoxylate cycle, β-oxidation and gluconeogenesis. Mutations in key enzymes such as ICL1 (glyoxylate cycle) and FOX2 (fatty acid β-oxidation) revealed that alternative carbon metabolic pathways are required for full virulence in C. albicans. These studies indicate C. albicans uses non-preferred carbon sources allowing its adaptation to microenvironments were nutrients are scarce. It has become apparent that the regulatory networks required for regulation of alternative carbon metabolism in C. albicans are considerably different from the Saccharomyces cerevisiae paradigm and appear more analogous to the Aspergillus nidulans systems. Well-characterized transcription factors in S. cerevisiae have no apparent phenotype or are missing in C. albicans. CTF1 was found to be a single functional homolog of the A. nidulans FarA/FarB proteins, which are transcription factors required for fatty acid utilization. Both FOX2 and ICL1 were found to be part of a large CTF1 regulon. To increase our understanding of how CTF1 regulates its target genes, including whether regulation is direct or indirect, the FOX2 and ICL1 promoter regions were analyzed using a combination of bioinformatics and promoter deletion analysis. To begin characterizing the FOX2 and ICL1 promoters, 5’ rapid amplification of cDNA ends (5’RACE) was used to identify two transcriptional initiation sites in FOX2 and one in ICL1. GFP reporter assays show FOX2 and ICL1 are rapidly expressed in the presence of alternative carbon sources. Both FOX2 and ICL1 harbor the CCTCGG sequence known to be bound by the Far proteins, hence rendering the motif as a putative CTF1 DNA binding element. In this study, the CCTCGG sequence was found to be essential for FOX2 regulation. However, this motif does not appear to be equally important for the regulation of ICL1. This study supports the notion that although C. albicans has diverged from the paradigms of model fungi, C. albicans has made specific adaptations to its transcription-based regulatory network that may contribute to its metabolic flexibility.

An Automatic Quantification and Registration Strategy to Create a Gene Expression Atlas of Zebrafish Embryogenesis

In order to properly understand and model the gene regulatory networks in animals development, it is crucial to obtain detailed measurements, both in time and space, about their gene expression domains. In this paper, we propose a complete computational framework to fulfill this task and create a 3D Atlas of the early zebrafish embryogenesis annotated with both the cellular localizations and the level of expression of different genes at different developmental stages. The strategy to construct such an Atlas is described here with the expression pattern of 5 different genes at 6 hours of development post fertilization.

Diseño y desarrollo de un modelo booleano probabilístico de regulación genética en el simulador de colonias bacterianas GRO

Resulta interesante comprender como microorganismos sencillos como la bacteria Escherichia coli poseen mecanismos no tan simples para responder al entorno en el que está gestionada por complicadas redes de regulación formadas por genes y proteínas, donde cada elemento de la red genética debe tomar parte en armonía, en el momento justo y la cantidad adecuada para dar lugar a la respuesta celular apropiada. La biología sintética es un nuevo área de la biología y la tecnología que fusiona la biolog ía molecular, la ingeniería genética y las herramientas computacionales, para crear sistemas biológicos con funcionalidades novedosas. Los sistemas creados sintéticamente son ya una realidad, y cada vez se acumulan más trabajos alrededor del mundo que muestran su factibilidad. En este campo no solo se hacen pequeñas modificaciones en la información genética, sino que también se diseñan, manipulan e introducen circuitos genéticos a los organismos. Actualmente, se hace un gran esfuerzo para construir circuitos genéticos formados por numerosos genes y caracterizar la interacción de los mismos con otras moléculas, su regulaci ón, expresión y funcionalidad en diferentes organismos. La mayoría de los proyectos de biología sintética que se han desarrollado hasta ahora, se basan en el conocimiento actual del funcionamiento de los organismos vivos. Sin embargo, la información es numerosa y creciente, por lo que se requiere de herramientas computacionales y matem áticas para integrar y hacer manejable esta gran cantidad de información. El simulador de colonias bacterianas GRO posee la capacidad de representar las dinámicas más simples del comportamiento celular, tales como crecimiento, división y comunicación intercelular mediante conjugación, pero carece de la capacidad de simular el comportamiento de la colonia en presencia de un circuito genético. Para ello, se ha creado un nuevo módulo de regulación genética que maneja las interaciones entre genes y proteínas de cada célula ejecutando respuestas celulares específicas. Dado que en la mayoría de los experimentos intervienen colonias del orden de 105 individuos, es necesario un módulo de regulación genética simplificado que permita representar de la forma más precisa posible este proceso en colonias de tales magnitudes. El módulo genético integrado en GRO se basa en una red booleana, en la que un gen puede transitar entre dos estados, on (expresado) o off (reprimido), y cuya transición viene dada por una serie de reglas lógicas.---ABSTRACT---It is interesting to understand how simple organisms such as Escherichia coli do not have simple mechanisms to respond to the environment in which they find themselves. This response is managed by complicated regulatory networks formed by genes and proteins, where each element of the genetic network should take part in harmony, at the right time and with the right amount to give rise to the appropriate cellular response. Synthetic biology is a new area of biology and technology that combines molecular biology, genetic engineering and computational tools to create biological systems with novel features. The synthetically created systems are already a reality, and increasingly accumulate work around the world showing their feasibility. In this field not only minor changes are made in the genetic information but also genetic circuits designed, manipulated and introduced into the organisms. Currently, it takes great effort to build genetic circuits formed by numerous genes and characterize their interaction with other molecules, their regulation, their expression and their function in different organisms. Most synthetic biology projects that have been developed so far are based on the current knowledge of the functioning of living organisms. However, there is a lot of information and it keeps accumulating, so it requires computational and mathematical tools to integrate and manage this wealth of information. The bacterial colonies simulator, GRO, has the ability to represent the simplest dynamics of cell behavior, such as growth, division and intercellular communication by conjugation, but lacks the ability to simulate the behavior of the colony in the presence of a genetic circuit. To this end, a new genetic regulation module that handles interactions between genes and proteins for each cell running specific cellular responses has been created. Since most experiments involve colonies of about 105 individuals, a simplified genetic module which represent cell dynamics as accurately and simply as possible is needed. The integrated genetic GRO module is based on a Boolean network, in which a gene can be in either of two states, on (expressed) or off (repressed), and whose transition is given by a set of logical rules.

Phylogenetic footprinting of transcription factor binding sites in proteobacterial genomes

Toward the goal of identifying complete sets of transcription factor (TF)-binding sites in the genomes of several gamma proteobacteria, and hence describing their transcription regulatory networks, we present a phylogenetic footprinting method for identifying these sites. Probable transcription regulatory sites upstream of Escherichia coli genes were identified by cross-species comparison using an extended Gibbs sampling algorithm. Close examination of a study set of 184 genes with documented transcription regulatory sites revealed that when orthologous data were available from at least two other gamma proteobacterial species, 81% of our predictions corresponded with the documented sites, and 67% corresponded when data from only one other species were available. That the remaining predictions included bona fide TF-binding sites was proven by affinity purification of a putative transcription factor (YijC) bound to such a site upstream of the fabA gene. Predicted regulatory sites for 2097 E.coli genes are available at http://www.wadsworth.org/resnres/bioinfo/.

Inhibition of the association of RNA polymerase II with the preinitiation complex by a viral transcriptional repressor.

Transcriptional repression is an important component of regulatory networks that govern gene expression. In this report, we have characterized the mechanisms by which the immediate early protein 2 (IE2 or IE86), a master transcriptional regulator of human cytomegalovirus, down-regulates its own expression. In vitro transcription and DNA binding experiments demonstrate that IE2 blocks specifically the association of RNA polymerase II with the preinitiation complex. Although, to our knowledge, this is the first report to describe a eukaryotic transcriptional repressor that selectively impedes RNA polymerase II recruitment, we present data that suggest that this type of repression might be widely used in the control of transcription by RNA polymerase II.

LPS regulates proinflammatory gene expression in macrophages by altering histone deacetylase expression

Bacterial LPS triggers dramatic changes in gene expression in macrophages. We show here that LPS regulated several members of the histone deacetylase (HDAC) family at the mRNA level in murine bone marrow-derived macrophages (BMM). LPS transiently repressed, then induced a number of HDACs (Hdac-4, 5, 7) in BMM, whereas Hdac-1 mRNA was induced more rapidly. Treatment of BMM with trichostatin A (TSA), an inhibitor of HDACs, enhanced LPS-induced expression of the Cox-2, Cxcl2, and Ifit2 genes. In the case of Cox-2, this effect was also apparent at the promoter level. Overexpression of Hdac-8 in RAW264 murine macrophages blocked the ability of LPS to induce Cox-2 mRNA. Another class of LPS-inducible genes, which included Ccl2, Ccl7, and Edn1, was suppressed by TSA, an effect most likely mediated by PU.1 degradation. Hence, HDACs act as potent and selective negative regulators of proinflammatory gene expression and act to prevent excessive inflammatory responses in macrophages.

Non-coding RNA

The term non-coding RNA (ncRNA) is commonly employed for RNA that does not encode a protein, but this does not mean that such RNAs do not contain information nor have function. Although it has been generally assumed that most genetic information is transacted by proteins, recent evidence suggests that the majority of the genomes of mammals and other complex organisms is in fact transcribed into ncRNAs, many of which are alternatively spliced and/or processed into smaller products. These ncRNAs include microRNAs and snoRNAs (many if not most of which remain to be identified), as well as likely other classes of yet-to-be-discovered small regulatory RNAs, and tens of thousands of longer transcripts (including complex patterns of interlacing and overlapping sense and antisense transcripts), most of whose functions are unknown. These RNAs (including those derived from introns) appear to comprise a hidden layer of internal signals that control various levels of gene expression in physiology and development, including chromatin architecture/epigenetic memory, transcription, RNA splicing, editing, translation and turnover. RNA regulatory networks may determine most of our complex characteristics, play a significant role in disease and constitute an unexplored world of genetic variation both within and between species.